Methods and compositions for modulation of sleep cycle

ABSTRACT

Methods and compositions for modulating the circadian rhythm of a subject are provided. In practicing the subject methods, modulation of the circadian rhythm of a subject is achieved by administering to the subject an effective amount of at least one of a glucocorticoid receptor antagonist, a CRH antagonist and an MR agonist. Also provided are kits for practicing the subject methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

Pursuant to 35 U.S.C. §119(e), this application claims priority to thefiling date of the U.S. Provisional Patent Application Ser. No.60/774,355 filed Feb. 17, 2006; the disclosure of which provisionalapplications is herein incorporated by reference.

GOVERNMENT RIGHTS

This invention was made with government support under federal grant nos.MH19938 awarded by National Institute of Mental Health. The UnitedStates Government may have certain rights in this invention.

INTRODUCTION

It is well established that living organisms have internal biologicalclocks which regulate activities such as their sleep/wake cycles. Thesebiological clocks are expressions of the effects of one or moreendogenous pacemakers thought to be located in the suprachiasmaticnuclei (SCN) of the hypothalamus. Biological rhythms are repetitivefluctuations in various biological signals over time, and are oftendriven by the biological clocks. A circadian rhythm occurs when theperiod of that fluctuation is about a day or about 24 hours. A circadianrhythm can manifest in terms of numerous biological variables andmeasures. Examples include the circadian rhythm to alertness, thecircadian rhythm to core body temperature, the circadian rhythm tocertain hormone production, the circadian rhythm to blood pressure, thecircadian rhythm to activity, the circadian rhythm of wake/sleep cycles,etc. Examples of hormonal circadian rhythms include those of cortisol,CRH and melatonin.

Different organisms have different activity cycles. Creatures which tendto be active during periods of daylight and inactive at night are termeddiurnal. Creatures which are active at night and sleep during the dayare referred to as nocturnal. In general, the phase or timing of naturalcircadian rhythms are entrained by and tend to follow the naturalsequence of daytime light and nighttime darkness which occurs as theearth rotates. Hence light has a chronobiotic effect, or phase shiftingeffect on the circadian rhythm and can assist with synchronization ofthe timing of the circadian rhythm.

On the other hand, there may be certain shapes to the waveform of eachof these different circadian rhythms. For example,.some waveforms may besinusoidal, linear, or a combination of several superimposed waveforms.In some cases, it may be useful to adjust the shape of the circadianwaveform to assist with nocturnal sleep. In some cases, the amplitude ofthe circadian rhythm amplitude may be flattened, its magnitude at thenadir may be increased, the entire waveform may be elevated inmagnitude, the time between acrophase and nadir may be shortened orprolonged, etc.

Under normal circumstances, the circadian rhythm of humans serves as auseful time regulator of various activities. However, some personssuffer from circadian rhythm phase irregularities such as advanced orCircadian Rhythm Sleep Disorder, Delayed Sleep Phase Type which suchdisorders result in an interference with maintenance of normal activitypattern. As a result such persons suffering from circadian rhythmirregularities and disorders experience disruptions in their sleeppatterns.

On the other hand, some suffer from disorders associated withalterations in the shape of the waveform of certain circadian rhythms.For example, in insomnia, the shape of the waveform of the circadianrhythm of cortisol may be elevated at certain times of night, comparedto controls (Vgontzas et al., 2001, J. Clin. Endocrinol. Metabl.,86(4):1489-1495; and Rodenbeck and Hajak, J Clin Psychiatry. 2001 June;62(6):453-63). An elevated nocturnal cortisol or ACTH at the nadir maybe a marker for increased HPA activity at night. As a result, suchcircadian waveform irregularities may disrupt sleep patterns.

In addition, in other instances, the internal circadian clock interfereswith desired adaptations to differing time schedules. For example, airtravelers who rapidly cross two or more time zones may find theirinternal circadian clocks out of phase with the day/night cycle at theirdestination, giving rise to the so-called “jet-lag” syndrome in whichthey suffer disruptions of their sleep patterns and diminished attentionspan and alertness until their inner biological clocks gradually adjustto local time. Shift workers, whose work schedules rotate among dayshift, night shift and the so-called “graveyard” shift, may experiencetransient internal temporal dissociation or a lack of synchronizationamong various bodily rhythms, and consequent difficulty in adjusting toshift changes. This can adversely affect worker productivity, and insome instances may raise safety concerns.

A general need exists for the regulation and control of the circadianrhythm of a subject, Regulation and control of the circadian rhythm of asubject would be beneficial for a number of diseases or disordersrelated to circadian rhythm irregularities, where control of thecircadian rhythm will contribute to treatment of a sleep disorder. Inaddition, control of the circadian rhythm of a subject would also bebeneficial in instances in which the internal circadian rhythminterferes with desired adaptations to differing time schedules.Accordingly, there continues to be a need for development of suchmethods.

SUMMARY

Methods and compositions for modulating the circadian rhythm of asubject are provided. In practicing the subject methods, modulation ofthe circadian rhythm of a subject is achieved by administering to thesubject an effective amount of at least one of a glucocorticoid receptorantagonist, a CRH antagonist and an MR agonist. Also provided are kitsfor practicing the subject methods.

Aspects of the invention include a method for modulating a circadianrhythm of a subject by administering to the subject an effective amountof a at least one of glucocorticoid receptor antagonist, a CRHantagonist and an MR agonist to modulate the circadian rhythm in thesubject. In one embodiment, the modulating results in phase shifting acircadian rhythm to improve the subject's sleep. In another embodiment,the modulating results in a change in the shape of the circadianwaveform to improve the subject's sleep. In another embodiment, themethod is a method for treating a subject for a sleep disorder. In suchembodiments the sleep disorder is insomnia, Circadian Rhythm SleepDisorder, Delayed Sleep Phase Type, Circadian Rhythm Sleep Disorder,Advanced Sleep Phase Type, Circadian Rhythm Sleep Disorder, IrregularSleep-Wake Type, Circadian Rhythm Sleep Disorder, Free-Running Type,short sleeper, long sleeper, obstructive sleep apnea. In otherembodiments the method is a method for treating inter-time zone travelinduced sleep disturbance (Circadian Rhythm Sleep Disorder, Jet LagType), e.g., Circadian Rhythm Sleep Disorder, Jet Lag Type. In yet otherembodiments the method is a method for treating Circadian Rhythm SleepDisorder, Shift Work Type. In certain embodiments the subject is amammal, such as a human.

In certain embodiments where the agent employed is a glucocorticoidreceptor antagonist, the glucocorticoid receptor antagonist includes asteroidal skeleton with at least one phenyl-containing moiety in the11-beta position of the steroidal skeleton. In further embodiments thephenyl-containing moiety in the 11-beta position of the skeleton is adimethylaminophenyl moiety. In yet further embodiments, theglucocorticoid receptor antagonist is mifepristone or is selected fromthe group consisting of RU009 and RU044.

Aspects of the invention further include methods for treating a sleepdisorder in a mammalian subject including administering to the subjectan effective amount of at least one of a glucocorticoid receptorantagonist, a CRH antagonist and an MR agonist to treat the sleepdisorder in the subject. In some embodiments, the sleep disorder isinsomnia. In other embodiments the sleep disorder is delayed sleep phasesyndrome. In yet other embodiments the sleep disorder is CircadianRhythm Sleep Disorder, Advanced Sleep Phase Type. In other embodimentsthe sleep disorder is shift-work sleep disorder. In other embodimentsthe sleep disorder is Circadian Rhythm Sleep Disorder, Jet Lag Type. Inother embodiments the sleep disorder is Circadian Rhythm Sleep Disorder,Irregular Sleep-Wake Type. In other embodiments the sleep disorder isCircadian Rhythm Sleep Disorder, Free-Running Type. In other embodimentsthe sleep disorder is short sleeper. In other embodiments, the sleepdisorder is long sleeper. In other embodiments the sleep disorder isobstructive sleep apnea. In such embodiments the subject may be a human.

Where employed, in certain embodiments the glucocorticoid receptorantagonist includes a steroidal skeleton with at least onephenyl-containing moiety in the 11-beta position of the steroidalskeleton. In further embodiments the phenyl-containing moiety in the11-beta position of the skeleton is a dimethylaminophenyl moiety. In yetfurther embodiments, the glucocorticoid receptor antagonist ismifepristone or is selected from the group consisting of RU009 andRU044.

Yet another feature of the present invention is a method for modulatingphase shifting of a circadian rhythm to accommodate an environmentallyimposed desired sleep cycle of a mammalian subject includingadministering to the subject an effective amount of at least one of aglucocorticoid receptor antagonist, a CRH antagonist and an MR agonistto modulate phase shifting of the sleep cycle of the subject. In someembodiments, the method is a method for treating a subject for aCircadian Rhythm Sleep Disorder, Shift Work Type. In other embodiments,the method is a method for treating a subject for inter-time zone travelinduced sleep disturbance (Circadian Rhythm Sleep Disorder, Jet LagType). In such embodiments the subject is a human.

Yet another aspect of the invention is a method for modulating arelative phase shift between two or more circadian rhythms, includingbetween cortisol and melatonin.

In certain embodiments the glucocorticoid receptor antagonist includes asteroidal skeleton with at least one phenyl-containing moiety in the11-beta position of the steroidal skeleton. In further embodiments thephenyl-containing moiety in the 11-beta position of the skeleton is adimethylaminophenyl moiety. In yet further embodiments, theglucocorticoid receptor antagonist is mifepristone or is selected fromthe group consisting of RU009 and RU044.

Yet another aspect of the present invention is a method of treating asleep disorder in a subject including identifying a subject sufferingfrom a sleep disorder; and administering to the subject an effectiveamount of at least one of a glucocorticoid receptor antagonist, a CRHantagonist and an MR agonist to treat the subject for the sleepdisorder. In some embodiments, the sleep disorder is insomnia. In otherembodiments, the sleep disorder is Circadian Rhythm Sleep Disorder,Delayed Sleep Phase Type. In yet other embodiments, the sleep disorderis Circadian Rhythm Sleep Disorder, Advanced Sleep Phase Type. In otherembodiments the sleep disorder is shift-work sleep disorder. In otherembodiments the sleep disorder is an inter-time zone travel inducedsleep disturbance (Circadian Rhythm Sleep Disorder, Jet Lag Type), e.g.,Circadian Rhythm Sleep Disorder, Jet Lag Type. In other embodiments thesleep disorder is Circadian Rhythm Sleep Disorder, Irregular Sleep-WakeType. In other embodiments the sleep disorder is Circadian Rhythm SleepDisorder, Free-Running Type. In other embodiments the sleep disorder isshort sleeper. In other embodiments, the sleep disorder is long sleeper.In other embodiments the sleep disorder is obstructive sleep apnea. Insuch embodiments the subject is a mammal, such as a human.

Yet another aspect of the present invention is a method of treating themetabolic or other endocrine consequences of a sleep disorder.Complications may include hypercortisolemia (i.e., as in sleep apnea)that may contribute to insomnia. Other endocrine complications of asleep disorder, such as obstructive sleep apnea, include metaboliccomplications such as hypercortisolemia induced insulin resistance.

In certain embodiments the glucocorticoid receptor antagonist includes asteroidal skeleton with at least one phenyl-containing moiety in the11-beta position of the steroidal skeleton. In further embodiments thephenyl-containing moiety in the 11-beta position of the skeleton is adimethylaminophenyl moiety. In yet further embodiments, theglucocorticoid receptor antagonist is mifepristone or is selected fromthe group consisting of RU009, RU044 ORG-34517, ORG-34850, andORG-34116.

Aspects of the present invention further include kits having a at leastone of a glucocorticoid receptor antagonist, CRH antagonist and MRagonist; and instructions for using the agent(s) to treat a sleepdisorder in a subject. In such embodiments where the agent is aglucocorticoid receptor antagonist, the glucocorticoid receptorantagonist includes a steroidal skeleton with at least onephenyl-containing moiety in the 11-beta position of the steroidalskeleton. In further embodiments the phenyl-containing moiety in the11-beta position of the skeleton is a dimethylaminophenyl moiety. In yetfurther embodiments, the glucocorticoid receptor antagonist ismifepristone or is selected from the group consisting of RU009 andRU044.

BRIEF DESCRIPTION OF THE FIGURES

The invention is best understood from the following detailed descriptionwhen read in conjunction with the accompanying drawings. It isemphasized that, according to common practice, the various features ofthe drawings are not to-scale. On the contrary, the dimensions of thevarious features may be arbitrarily expanded or reduced for clarity.Included in the drawings are the following figures:

FIG. 1 is a graph showing relative change in polysomnogram measures oftreatment group to placebo group. The scores show the difference betweenfive days following treatment with mifepristone compared to baseline andtwo weeks following discontinuation of mifepristone administrationcompared to baseline. Polysomnogram abbreviations: WASO=wake after sleeponset; % S1=percentage stage 1 sleep; % S2=percentage stage 2 sleep; %S3=percentage stage 3 sleep; % S4=percentage stage 4 sleep; %REM=percentage REM sleep; NUAW=number of awakenings; TST=total combinedminutes of sleep; S3 Lat (min)=latency to onset of stage 3 sleep;SEB=total combined minutes of stage 2, 3 and 4 per total sleep minutes;SEC=total combined minutes of stage 3, 4 per total sleep minutes; %NEM=sum of % S3 and % S4; REM lat=minutes to first epoch of REM aftersleep onset; REM density=# rapid eye movements per minutes of REM sleep;and Arousal index=computerized measure high frequency bands/minutessleep.

FIG. 2 is a graph showing absolute change in polysomnogram measures oftreatment group. The scores show the difference between five daysfollowing treatment with mifepristone compared to baseline and two weeksfollowing discontinuation of mifepristone administration compared tobaseline. Abbreviations are the same as in FIG. 1.

FIG. 3 is a series of graphs showing overnight melatonin levels in dimlights in chronic insomnia subjects that have been administered either aplacebo (Panel A) or mifepristone (Panel B). The graphs provide timepoints for measurements prior to administration, five days followingadministration, and two weeks following discontinuation ofadministration of either placebo (Panel A) or mifepristone (Panel B).

FIG. 4 is a series of graphs showing overnight cortisol (Panel A) andACTH (Panel B) levels in chronic insomnia subjects administered eithermifepristone or placebo. The longitudinal date of the graphs is dividedinto three sections: first section=prior to treatment; secondsection=−five days following administration; third section=two weeksfollowing discontinuation of administration.

FIG. 5 is a graph showing overnight cortisol levels in healthy controlsubjects that have been administered mifepristone. Diamonds showmeasurements prior to administration of mifepristone and squares showmeasurements two days after administration of mifepristone.

FIGS. 6 to 8 provide graphs of results from various studies reported inExample 3, below.

DEFINITIONS

The term “treating” refers to any indicia of success in the treatment oramelioration of an injury, pathology or condition, including anyobjective or subjective parameter such as abatement; remission;diminishing of symptoms or making the injury, pathology or conditionmore tolerable to the subject; slowing in the rate of degeneration ordecline; making the final point of degeneration less debilitating; orimproving a subject's physical or mental well-being. The treatment oramelioration of symptoms can be based on objective or subjectiveparameters; including the results of a physical examination and/orevaluation of sleep patterns.

The term “glucocorticoid receptor antagonist” refers to any compositionor compound which partially or completely inhibits (antagonizes) thebinding of a glucocorticoid receptor (GR) agonist, such as cortisol, orcortisol analogs, synthetic or natural, to a GR. A “glucocorticoidreceptor antagonist” also refers to any composition or compound whichinhibits any biological response associated with the binding of a GR toan agonist.

The term “glucocorticoid receptor” (“GR”) refers to a family ofintracellular receptors also referred to as the cortisol receptor, whichspecifically bind to cortisol and/or cortisol analogs. The term includesisoforms of GR, recombinant GR and mutated GR.

The term “cortisol” refers to a family of compositions also referred tohydrocortisone, and any synthetic or natural analogues thereof.

The term “mifepristone” refers to a family of compositions also referredto as RU486, or RU38.486, or17-beta-hydroxy-11-beta-(4-dimethyl-aminophenyl)-17-alpha-(1-propynyl)-estra-4,9-dien-3-one),or11-beta-(4dimethylaminophenyl)-17-beta-hydroxy-17-alpha-(1-propynyl)-estra-4,9-dien-3-one),or analogs thereof, which bind to the glucocorticoid receptor, typicallywith high affinity, and inhibit the biological effectsinitiated/mediated by the binding of any cortisol or cortisol analogueto a receptor. Chemical names for RU-486 vary; for example, RU486 hasalso been termed:11B-[p-(Dimethylamino)phenyl)-17B-hydroxy-17-(1-propynyl)-estra-4,9-dien-3-one;11B-(4-dimethyl-aminophenyl)-17B-hydroxy-17A-(prop-1-ynyl)-estra-4,9-dien-3-one;17B-hydroxy-11B-(4-dimethylaminophenyl-1)-17A-(propynyl-1)-estra-4,9-diene-3-one;17B-hydroxy-11B-(4-dimethylaminophenyl-1)-17A-(propynyl-1)-E;(11B,17B)-11[4-dimethylamino)-phenyl]-17-hydroxy-17-(1-propynyl)estra-4,9-dien-3-one;and11B-[4-(N,N-dimethylamino)phenyl]-17A-(prop-1-ynyl)-D-4,9-estradiene-17B-ol-3-one.

DETAILED DESCRIPTION

Methods and compositions for modulating the circadian rhythm of asubject are provided. In practicing the subject methods, modulation ofthe circadian rhythm of a subject is achieved by administering to thesubject an effective amount of a glucocorticoid receptor antagonist.Also provided are kits for practicing the subject methods.

Before the present invention is described further, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Certain ranges are presented herein with numerical values being precededby the term “about.” The term “about” is used herein to provide literalsupport for the exact number that it precedes, as well as a number thatis near to or approximately the number that the term precedes. Indetermining whether a number is near to or approximately a specificallyrecited number, the near or approximating unrecited number may be anumber which, in the context in which it is presented, provides thesubstantial equivalent of the specifically recited number.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference to disclose and describe the methodsand/or materials in connection with which the publications are cited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood to one of ordinary skill in the art to which thisinvention belongs.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

In further describing the invention, representative methods arediscussed first in greater detail, followed by a review ofrepresentative applications for the methods, as well as kits that finduse in practicing the subject methods.

Methods

As summarized above, the subject invention provides a method formodulating a circadian rhythm of a subject. By “circadian rhythm” ismeant the daily, i.e., 24 hour, Hypothalamic-Pituitary Adrenal (HPA)axis cycle of cortisol, Adrenocorticotropic Hormone (ACTH) orcorticotropin-releasing hormone (CRH), melatonin, core body temperature.By “circadian rhythm of the activity cycle” is meant the 24 hour cycleof activity and rest patterns. By “circadian rhythm of a sleep cycle” ismeant the 24 hour cycle of sleep versus wake state, where a normal sleepcycle is characterized by six states, consisting of an awake stage(stage W), and sleep stage, which stage includes light sleep (stages 1and 2), slow wave sleep (stages 3 and 4) and rapid-eye movement sleep(REM) stage. During such a normal sleep cycle, the greatest portion ofslow-wave sleep (SWS) occurs in the first part of the night, and thegreatest portion of REM occurs in the second half of the night. As usedherein “modulating” means changing, including delaying and progressing,the waveform shape, including the amplitude, etc., of the circadianrhythm of a subject. As such, in some embodiments the modulating mayresult in phase-advancing the circadian rhythm, wherein the circadianrhythm of the sleep cycle is advanced from an awake state to a sleepstate. -In other embodiments the modulating may result in phase-delayingthe circadian rhythm, wherein the circadian rhythm of the sleep cycle isdelayed from progressing from an awake state to a sleep state.

In such embodiments, phase shifting, e.g., phase-advancement orphase-delay, in the circadian rhythm can be monitored based onmeasurements of the subject's circadian markers that can reasonablypredict the endogenous circadian pacemaker. Such circadian markersinclude, but are not limited to, the following: body core temperature,which decreases as the sleep cycle advances from the awake stage to thesleep stage, sleep latency (i.e., the time it takes to fall asleep),natural wake time, timing of the nadir of cortisol production, timing ofthe acrophase of cortisol production, timing of the Dim Light MelatoninOnset (DLMO), timing of DIM Light Melatonin Offset, timing of onset ofREM latency relative to sleep onset (REM latency). In such methods, thephase of a circadian rhythm of a subject is modulated by administeringto the subject an effective amount of a glucocorticoid receptorantagonist in a manner sufficient to modulate the circadian rhythm ofthe subject.

In other embodiments, the modulating effect results in adjusting theshape of the circadian rhythm waveform to improve sleep. In suchmethods, the waveform shape can be monitored or measured by othercharacteristics. Such characteristics include, but are not limited to,height of nadir, amplitude of waveform, height of acrophase, mesor,distance between the acrophase and nadir, area under the curve.

A variety of subjects are treatable according to the subject methods.Generally such hosts are “mammals” or “mammalian,” where these terms areused broadly to describe organisms which are within the class mammalia,including the orders carnivore (e.g., dogs and cats), rodentia (e.g.,mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees,and monkeys). In many embodiments, the subjects will be humans.

The invention provides for methods of modulating a circadian rhythm of asubject by administering to the subject an effective amount of at leastone of a glucocorticoid receptor (GR) antagonist, a CRH antagonist and aMR agonist to modulate the circadian rhythm of the subject. In certainembodiments, only one of these agents is employed. In certainembodiments, two or these agents are employed. In certain embodiments,three of these agents are employed. Antagonists of GR activity utilizedin embodiments of the methods of the invention are well described in thescientific and patent literature. A few illustrative examples are setforth below. Likewise, CRH antagonists utilized in embodiments of themethods of the invention are well described in the scientific and patentliterature. A few illustrative examples are set forth below. Likewise,MR agonists utilized in embodiments of the methods of the invention arewell described in the scientific and patent literature. A fewillustrative examples are set forth below.

Suitable for use in embodiments of the subject methods are steroidalanti-glucocorticoids as GR antagonists. Steroidal antiglucocorticoidscan be obtained by modification of the basic structure of glucocorticoidagonists, i.e., varied forms of the steroid backbone. The structure ofcortisol, which binds the GR, can be modified in a variety of ways. Thetwo most commonly known classes of structural modifications of thecortisol steroid backbone to create glucocorticoid antagonists includemodifications of the 11-beta hydroxy group and modification of the17-beta side chain (see, e.g., Lefebvre (1989) J. Steroid Biochem.33:557-563).

Glucocorticoid agonists with modified steroidal backbones comprisingremoval or substitution of the 11-beta hydroxy group are administered inone embodiment of the invention. This class includes naturalantiglucocorticoids, including cortexolone, progesterone andtestosterone derivatives, and synthetic compositions, such asmifepristone (Lefebvre, et al. (1989) Ibid). An exemplary embodiment ofthe invention includes all 11-beta-aryl steroid backbone derivativesbecause these compounds are devoid of progesterone receptor (PR) bindingactivity (Agarwal (1987) FEBS 217:221-226). Another exemplary embodimentcomprises an 11-beta phenyl-aminodimethyl steroid backbone derivative,i.e., mifepristone, which is both an effective anti-glucocorticoid andanti-progesterone agent. These compositions act as reversibly-bindingsteroid receptor antagonists. For example, when bound to a 1-betaphenyl-aminodimethyl steroid, the steroid receptor is maintained in aconformation that cannot bind its natural ligand, such as cortisol inthe case of GR (Cadepond (1997), supra).

Synthetic 11-beta phenyl-aminodimethyl steroids include mifepristone,also known as RU486, or 17-beta-hydrox-11-beta-(4-dimethyl-aminophenyl)17-alpha-(1-propynyl)estra-4,9-dien-3-one). Mifepristone has been shownto be a powerful antagonist of both the progesterone and glucocorticoid(GR) receptors. Another 11-beta phenyl-aminodimethyl steroids shown tohave GR antagonist effects includes RU009 (RU39.009),11-beta-(4-dimethyl-aminoethoxyphenyl)-17-alpha-(propynyl-17beta-hydroxy-4,9-estradien-3-one)(see Bocquel (1993) J. Steroid Biochem. Molec. Biol. 45:205-215).Another GR antagonist related to RU486 is RU044 (RU43.044)17-beta-hydrox-17-alpha-19-(4-methyl-phenyl)-androsta-4,9(11)-dien-3-one)(Bocquel (1993) supra). See also Teutsch (1981) Steroids 38:651-665;U.S. Pat. Nos. 4,386,085 and 4,912,097. Other exemplary glucocorticoidreceptor antagonists include, but are not limited to, ORG-34517,ORG-34850, and ORG-34116.

One embodiment includes compositions containing the basic glucocorticoidsteroid structure which are irreversible anti-glucocorticoids. Suchcompounds include alpha-keto-methanesulfonate derivatives of cortisol,including cortisol-21-mesylate (4-pregnene-11-beta, 17-alpha,21-triol-3,20-dione-21-methane-sulfonate and dexamethasone-21-mesylate(16-methyl-9alpha-fluoro-1,4-pregnadiene-11beta, 17-alpha,21-triol-3,20-dione-21-methane-sulfonate). See Simons (1986) J. SteroidBiochem. 24:25-32 (1986); Mercier (1986) J. Steroid Biochem. 25:11-20;U.S. Pat. No. 4,296,206.

Steroidal antiglucocorticoids which can be obtained by variousstructural modifications of the 17-beta side chain are also used in themethods of the invention. This class includes syntheticantiglucocorticoids such as dexamethasone-oxetanone, various17,21-acetonide derivatives and 17-beta-carboxamide derivatives ofdexamethasone (Lefebvre (1989) supra; Rousseau (1979) Nature279:158-160).

GR antagonists used in the various embodiments of the invention includeany steroid backbone modification which effects a biological responseresulting from a GR-agonist interaction. Steroid backbone antagonistscan be any natural or synthetic variation of cortisol, such as adrenalsteroids missing the C-19 methyl group, such as19-nordeoxycorticosterone and 19-norprogesterone (Wynne (1980)Endocrinology 107:1278-1280).

In general, the 11-beta side chain substituent, and particularly thesize of that substituent, can play a key role in determining the extentof a steroid's antiglucocorticoid activity. Substitutions in the A ringof the steroid backbone can also be important. 17-hydroxypropenyl sidechains generally decrease antiglucocorticoidal activity in comparison to17-propinyl side chain containing compounds.

Non-steroidal glucocorticoid antagonists are also used in the subjectmethods of the invention. These include synthetic mimetics and analogsof proteins, including partially peptidic, pseudopeptidic andnon-peptidic molecular entities. For example, oligomeric peptidomimeticsuseful in the invention include (alpha-beta-unsaturated)peptidosulfonamides, N-substituted glycine derivatives, oligocarbamates, oligo urea peptidomimetics, hydrazinopeptides, oligosulfonesand the like (see, e.g., Amour (1994) Int. J. Pept. Protein Res.43:297-304; de Bont (1996) Bioorganic & Medicinal Chem. 4:667-672). Thecreation and simultaneous screening of large libraries of syntheticmolecules can be carried out using well-known techniques incombinatorial chemistry, for example, see van Breemen (1997) Anal Chem69:2159-2164; Lam (1997) Anticancer Drug Des 12:145-167 (1997). Designof peptidomimetics specific for GR can be designed using computerprograms in conjunction with combinatorial chemistry (combinatoriallibrary) screening approaches (Murray (1995) J. of Computer-Aided Molec.Design 9:381-395); Bohm (1996) J. of Computer-Aided Molec. Design10:265-272). Such “rational drug design” can help develop peptideisomerics and conformers including cycloisomers, retro-inverso isomers,retro isomers and the like (as discussed in Chorev (1995) Tib Tech13:438-445).

Because any GR antagonist can be used for the subject methods of theinvention, in addition to the compounds and compositions describedabove, additional useful GR antagonists can be determined by the skilledartisan. A variety of such routine, well-known methods can be used andare described in the scientific and patent literature. They include invitro and in vivo assays for the identification of additional GRantagonists. A few illustrative examples are described below.

One assay that can be used to identify a GR antagonist of the inventionmeasures the effect of a putative GR antagonist on tyrosineamino-transferase activity in accordance with the method of Granner(1970) Meth. Enzymol. 15:633. This analysis is based on measurement ofthe activity of the liver enzyme tyrosine amino-transferase (TAT) incultures of rat hepatoma cells (RHC). TAT catalyzes the first step inthe metabolism of tyrosine and is induced by glucocorticoids (cortisol)both in liver and hepatoma cells. This activity is easily measured incell extracts. TAT converts the amino-group of tyrosine to 2-oxoglutaricacid. P-hydroxyphenylpyruvate is also formed. It can be converted to themore stable p-hydroxybenzaldehyde in an alkaline solution andquantitated by absorbance at 331 nm. The putative GR antagonist isco-administered with cortisol to whole liver, in vivo or ex vivo, orhepatoma cells or cell extracts. A compound is identified as a GRantagonist when its administration decreases the amount of induced TATactivity, as compared to control (i.e., only cortisol or GR agonistadded) (see also Shirwany (1986) “Glucocorticoid regulation of hepaticcytosolic glucocorticoid receptors in vivo and its relationship toinduction of tyrosine aminotransferase,” Biochem. Biophys. Acta886:162-168).

Further illustrative of the many assays which can be used to identifycompositions utilized in the methods of the invention, in addition tothe TAT assay, are assays based on glucocorticoid activities in vivo.For example, assays that assess the ability of a putative GR antagonistto inhibit uptake of ³H-thymidine into DNA in cells which are stimulatedby glucocorticoids can be used. Alternatively, the putative GRantagonist can complete with ³H-dexamethasone for binding to a hepatomatissue culture GR (see, e.g., Choi (1992) “Enzyme induction andreceptor-binding affinity of steroidal 20-carboxamides in rat hepatomatissue culture cells,” Steroids 57:313-318). As another example, theability of a putative GR antagonist to block nuclear binding of³H-dexamethasone-GR complex can be used (Alexandrova (1992) “Duration ofantagonizing effect of RU486 on the agonist induction of tyrosineaminotransferase via glucocorticoid receptor,” J. Steroid Biochem. Mol.Biol. 41:723-725). To further identify putative GR antagonists, kineticassays able to discriminate between glucocorticoid agonists andantagonists by means of receptor-binding kinetics can also be used (asdescribed in Jones (1982) Biochem J. 204:721-729).

In another illustrative example, the assay described by Daune (1977)Molec. Pharm. 13:948-955, and in U.S. Pat. No. 4,386,085, can be used toidentify anti-glucocorticoid activity. Briefly, the thymocytes ofsurrenalectomized rats are incubated in nutritive medium containingdexamethasone with the test compound (the putative GR antagonist) atvarying concentrations. ³H-uridine is added to the cell culture, whichis further incubated, and the extent of incorporation of radiolabel intopolynucleotide is measured. Glucocorticoid agonists decrease the amountof ³H-uridine incorporated. Thus, a GR antagonist will oppose thiseffect.

For additional compounds that can be utilized in the methods of theinvention and methods of identifying and making such compounds, see U.S.Pat. No. 4,296,206 (see above); U.S. Pat. No. 4,386,085 (see above);U.S. Pat. Nos. 4,447,424; 4,477,445; 4,519,946; 4,540,686; 4,547,493;4,634,695; 4,634,696; 4,753,932; 4,774,236; 4,808,710; 4,814,327;4,829,060; 4,861,763; 4,912,097; 4,921,638; 4,943,566; 4,954,490;4,978,657; 5,006,518; 5,043,332; 5,064,822; 5,073,548; 5,089,488;5,089,635; 5,093,507; 5,095,010; 5,095,129; 5,132,299; 5,166,146;5,166,199; 5,173,405; 5,276,023; 5,380,839; 5,348,729; 5,426,102;5,439,913; and 5,616,458; and WO 96/19458, which describes non-steroidalcompounds which are high-affinity, highly selective modulators(antagonists) for steroid receptors, such as 6-substituted-1,2-dihydroN-1 protected quinolines.

Antiglucocorticoids, such as mifepristone, are formulated aspharmaceuticals to be used in the subject methods of the invention.Routine means to determine GR antagonist drug regimens and formulationsto practice the methods of the invention are well described in thepatent and scientific literature, and some illustrative examples are setforth below.

The GR antagonists used in the methods of the invention can beadministered by any means known in the art, e.g., parenterally,topically, orally, or by local administration, such as by aerosol ortransdermally. The methods of the invention provide for prophylacticand/or therapeutic treatments. The GR antagonists as pharmaceuticalformulations can be administered in a variety of unit dosage formsdepending-upon the condition, disease or disorder, the generalmedical-condition of each subject, the resulting preferred method ofadministration and the like. Details on techniques for formulation andadministration are well described in the scientific and patentliterature, see, e.g., the latest edition of Remington's PharmaceuticalSciences, Maack Publishing Co, Easton Pa. (“Remington's”).

GR antagonist pharmaceutical formulations can be prepared according toany method known to the art for the manufacture of pharmaceuticals. Suchdrugs can contain sweetening agents, flavoring agents, coloring agentsand preserving agents. Any GR antagonist formulation can be admixturedwith nontoxic pharmaceutically acceptable excipients which are suitablefor manufacture.

Pharmaceutical formulations for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art inappropriate and suitable dosages. Such carriers enable thepharmaceutical formulations to be formulated in unit dosage forms astablets, pills, powder, dragees, capsules, liquids, lozenges, gels,syrups, slurries, suspensions, etc., suitable for ingestion by thesubject. Pharmaceutical preparations for oral use can be obtainedthrough combination of GR antagonist compounds with a solid excipient,optionally grinding a resulting mixture, and processing the mixture ofgranules, after adding suitable additional compounds, if desired, toobtain tablets or dragee cores. Suitable solid excipients arecarbohydrate or protein fillers include, but are not limited to sugars,including lactose, sucrose, mannitol, or sorbitol; starch from corn,wheat, rice, potato, or other plants; cellulose such as methylcellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; and gums including arabic and tragacanth; aswell as proteins such as gelatin and collagen. If desired,disintegrating or solubilizing agents may be added, such as thecross-linked polyvinyl pyrrolidone, agar, alginic acid, or a saltthereof, such as sodium alginate.

Dragee cores are provided with suitable coatings such as concentratedsugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound (i.e., dosage). Pharmaceutical preparations of theinvention can also be used orally using, for example, push-fit capsulesmade of gelatin, as well as soft, sealed capsules made of gelatin and acoating such as glycerol or sorbitol. Push-fit capsules can contain GRantagonist mixed with a filler or binders such as lactose or starches,lubricants such as talc or magnesium stearate, and, optionally,stabilizers. In soft capsules, the GR antagonist compounds may bedissolved or suspended in suitable liquids, such as fatty oils, liquidparaffin, or liquid polyethylene glycol with or without stabilizers.

Aqueous suspensions of the invention contain a GR antagonist inadmixture with excipients suitable for the manufacture of aqueoussuspensions. Such excipients include a suspending agent, such as sodiumcarboxymethylcellu lose, methylcellu lose, hydroxypropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia,and dispersing or wetting agents such as a naturally occurringphosphatide (e.g., lecithin), a condensation product of an alkyleneoxide with a fatty acid (e.g., polyoxyethylene stearate), a condensationproduct of ethylene oxide with a long chain aliphatic alcohol (e.g.,heptadecaethylene oxycetanol), a condensation product of ethylene oxidewith a partial ester derived from a fatty acid and a hexitol (e.g.,polyoxyethylene sorbitol mono-oleate), or a condensation product ofethylene oxide with a partial ester derived from fatty acid and ahexitol anhydride (e.g., polyoxyethylene sorbitan mono-oleate). Theaqueous suspension can also contain one or more preservatives such asethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one ormore flavoring agents and one or more sweetening agents, such assucrose, aspartame or saccharin. Formulations can be adjusted forosmolarity.

Oil suspensions can be formulated by suspending a GR antagonist in avegetable oil, such as arachis oil, olive oil, sesame oil or coconutoil, or in a mineral oil such as liquid paraffin; or a mixture of these.The oil suspensions can contain a thickening agent, such as beeswax,hard paraffin or cetyl alcohol. Sweetening agents can be added toprovide a palatable oral preparation, such as glycerol, sorbitol orsucrose. These formulations can be preserved by the addition of anantioxidant such as ascorbic acid. As an example of an injectable oilvehicle, see Minto (1997) J. Pharmacol Exp. Ther. 281:93-102. Thepharmaceutical formulations of the invention can also be in the form ofoil-in-water emulsions. The oily phase can be a vegetable oil or amineral oil, described above, or a mixture of these. Suitableemulsifying agents include naturally-occurring gums, such as gum acaciaand gum tragacanth, naturally occurring phosphatides, such as soybeanlecithin, esters or partial esters derived from fatty acids and hexitolanhydrides, such as sorbitan mono-oleate, and condensation products ofthese partial esters with ethylene oxide, such as polyoxyethylenesorbitan mono-oleate. The emulsion can also contain sweetening agentsand flavoring agents, as in the formulation of syrups and elixirs. Suchformulations can also contain a demulcent, a preservative, or a coloringagent.

Dispersible powders and granules of the invention suitable forpreparation of an aqueous suspension by the addition of water can beformulated from a GR antagonist in admixture with a dispersing,suspending and/or wetting agent, and one or more preservatives. Suitabledispersing or wetting agents and suspending agents are exemplified bythose disclosed above. Additional excipients, for example, sweetening,flavoring and coloring agents, can also be present.

The GR antagonists of this invention can also be administered in theform of suppositories for rectal administration of the drug. Theseformulations can be prepared by mixing the drug with a suitablenon-irritating excipient which is solid at ordinary temperatures butliquid at the rectal temperatures and will therefore melt in the rectumto release the drug. Such materials are cocoa butter and polyethyleneglycols.

The GR antagonists of this invention can also be administered by inintranasal, intraocular, intravaginal, and intrarectal routes includingsuppositories, insufflation, powders and aerosol formulations (forexamples of steroid inhalants, see Rohatagi (1995) J. Clin. Pharmacol.35:1187-1193; Tjwa (1995) Ann. Allergy Asthma Immunol. 75:107-111).

The GR antagonists of the invention can be delivered by transdermally,by a topical route, formulated as applicator sticks, solutions,suspensions, emulsions, gels, creams, ointments, pastes, jellies,paints, powders, and aerosols.

The GR antagonists of the invention can also be delivered asmicrospheres for slow release in the body. For example, microspheres canbe administered via intradermal injection of drug (e.g.,mifepristone)-containing microspheres, which slowly releasesubcutaneously (see Rao (1995) J. Biomater Sci. Polym. Ed. 7:623-645; asbiodegradable and injectable gel formulations, see, e.g., Gao (1995)Pharm. Res. 12:857-863 (1995); or, as microspheres for oraladministration, see, e.g., Eyles (1997) J. Pharm. Pharmacol.49:669-674). Both transdermal and intradermal routes afford constantdelivery for weeks or months.

The GR antagonist pharmaceutical formulations of the invention can beprovided as a salt and can be formed with many acids, including but notlimited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic,succinic, etc. Salts tend to be more soluble in aqueous or otherprotonic solvents that are the corresponding free base forms. In othercases, the preferred preparation may be a lyophilized powder in 1 mM-50mM histidine, 0.1%-2% sucrose, 2%-7% mannitol at a pH range of 4.5 to5.5, that is combined with buffer prior to use.

In another embodiment, the GR antagonist formulations of the inventionare useful for parenteral administration, such as intravenous (IV)administration or administration into a body cavity or lumen of anorgan. The formulations for administration will commonly comprise asolution of the GR antagonist (e.g., mifepristone) dissolved in apharmaceutically acceptable carrier. Among the acceptable vehicles andsolvents that can be employed are water and Ringer's solution, anisotonic sodium chloride. In addition, sterile fixed oils canconventionally be employed as a solvent or suspending medium. For thispurpose any bland fixed oil can be employed including synthetic mono- ordiglycerides. In addition, fatty acids such as oleic acid can likewisebe used in the preparation of injectables. These solutions are sterileand generally free of undesirable matter. These formulations may besterilized by conventional, well known sterilization techniques. Theformulations may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions such aspH adjusting and buffering agents, toxicity adjusting agents, e.g.,sodium acetate, sodium chloride, potassium chloride, calcium chloride,sodium lactate and the like. The concentration of GR antagonist in theseformulations can vary widely, and will be selected primarily based onfluid volumes, viscosities, body weight, and the like, in accordancewith the particular mode of administration selected and the subject'sneeds. For IV administration, the formulation can be a sterileinjectable preparation, such as a sterile injectable aqueous oroleaginous suspension. This suspension can be formulated according tothe known art using those suitable dispersing or wetting agents andsuspending agents. The sterile injectable preparation can also be asterile injectable solution or suspension in a nontoxicparenterally-acceptable diluent or solvent, such as a solution of1,3-butanediol.

In another embodiment, the GR antagonist formulations of the inventioncan be delivered by the use of liposomes which fuse with the cellularmembrane or are endocytosed, i.e., by employing ligands attached to theliposome, or attached directly to the oligonucleotide, that bind tosurface membrane protein receptors of the cell resulting in endocytosis.By using liposomes, particularly where the liposome surface carriesligands specific for target cells, or are otherwise preferentiallydirected to a specific organ, one can focus the delivery of the GRantagonist into the target cells in vivo See, e.g., Al-Muhammed (1996)J. MicroencapsuL 13:293-306; Chonn (1995) Curr. Opin. Biotechnol.6:698-708; Ostro (1989) Am. J. Hosp. Pharm. 46:1576-1587.

The amount of GR antagonist adequate to modulate the circadian rhythm ofa subject is defined as a “therapeutically effective dose.” The dosageschedule and amounts effective for this use, i.e., the “dosing regimen,”will depend upon a variety of factors, including the stage of thedisease or condition, the severity of the disorder or condition, thegeneral state of the subject's health, the subject's physical status,age and the like. In calculating the dosage regimen for a subject, themode of administration also is taken into consideration.

The dosage regimen also takes into consideration pharmacokineticsparameters well known in the art, i.e., the GR antagonists' rate ofabsorption, bioavailability, metabolism, clearance, and the like (see,e.g., Hidalgo-Aragones (1996) J. Steroid Biochem. Mol. Biol. 58:611-617;Groning (1996) Pharmazie 51:337-341; Fotherby (1996) Contraception54:59-69; Johnson (1995) J. Pharm. Sci. 84:1144-1146; Rohatagi (1995)Pharmazie 50:610-613; Brophy (1983) Eur. J Clin. Pharmacol. 24:103-108;the latest Remington's, supra). For example, in one study, less than0.5% of the daily dose of mifepristone was excreted in the urine; thedrug bound extensively to circulating albumin (see Kawai (1989) supra).The state of the art allows the clinician to determine the dosageregimen for each individual subject, GR antagonist and disease orcondition treated. As an illustrative example, the guidelines providedbelow for mifepristone can be used as guidance to determine the dosageregiment, i.e., dose schedule and dosage levels, of any GR antagonistadministered when practicing the methods of the invention.

Single or multiple administrations of GR antagonist formulations can beadministered depending on the dosage and frequency as required andtolerated by the subject. The formulations should provide a sufficientquantity of active agent, e.g., mifepristone, to effectively modulatethe circadian rhythm of a subject. Thus, one typical pharmaceuticalformulation for oral administration of a GR antagonist, such asmifepristone, is administered in a daily amount of between about 2 toabout 20 mg per kilogram of body weight per day, such as in a dailyamount of between about 2 to about 12 mg per kilogram of body weight perday, including in a daily amount of between about 2 to about 9 mg perkilogram of body weight per day.

Lower dosages can be used, particularly when the drug is administered toan anatomically secluded site, in contrast to administration orally,into the blood stream, into a body cavity or into a lumen of an organ.Substantially higher dosages can be used in topical administration.Actual methods for preparing parenterally administrable GR antagonistformulations will be known or apparent to those skilled in the art andare described in more detail in such publications as Remington's, supra.See also Nieman, In “Receptor Mediated Antisteroid Action,” Agarwal, etal., eds., De Gruyter, New York (1987).

After a pharmaceutical comprising a GR antagonist of the invention hasbeen formulated in an acceptable carrier, it can be placed in anappropriate container and labeled for use in the subject methods. Foradministration of GR antagonist, such labeling would include, e.g.,instructions concerning the amount, frequency and method ofadministration.

As summarized above, in some embodiments, the subject method ofmodulating the circadian rhythm of a subject is achieved byadministering to the subject an effective amount of a GR antagonist andan effective amount of an additional compound, such as a corticotropinreleasing hormone (CRH) antagonist or a mineralcorticoid receptor (MR)agonist. In other embodiments the subject method of modulating thecircadian rhythm of a subject is achieved by administering to thesubject an effective amount of a CRH antagonist. In yet otherembodiments, subject method of modulating the circadian rhythm of asubject is achieved by administering to the subject an effective amountof a MR agonist.

CRH antagonists suitable for use with the subject invention include, butare not limited to the following, antalarmnin, R-121919, astressin, andthe like.

MR agonist suitable for use with the subject invention include, but arenot limited to the following, deoxycorticosterone, spironolactone,fludrocortisone, aldosterone, progesterone, and the like.

In the above embodiments where CRH antagonists and/or MR agonists areemployed, these agents can be administered using any convenientformulation and protocol, such as the protocols described above inconnection with the administration of GR antagonists.

Modulating of the circadian rhythm of a subject can be readily evaluatedby methods apparent to one skilled in the art. Such methods ofevaluating whether the circadian rhythm of a subject has been modulatedinclude determining whether the subject exhibits an increased capabilityof controlling initiation of the sleep stage of the sleep cycle, orcontrolling the natural termination of the sleep stage of the sleepcycle or physiologically adapting to an environmentally imposed changein sleep cycle. Control of initiation of the sleep stage means thesubject is capable of initiating the sleep stage of the sleep cycle at aparticular desired time point. As such, control of initiation of thesleep stage includes the ability by the subject to initiate the sleepstage of the sleep cycle at a particular time, where such time isearlier than the usual expected time, i.e., occurring before the usualexpected time, where such time is at least about 30 minutes to about 12hours, such as at least about 2 to about 10 hours, including at leastabout 3 to about 7 hours before the usual expected time. Control ofinitiation of the sleep stage also includes the ability by the subjectto initiate the sleep stage of the sleep cycle at a particular time,where such time is later than the usual expected time, i.e., occurringafter the usual expected time, where such time is at least about 30minutes to about 12 hours, such as at least about 2 to about 10 hours,including at least about 3 to about 7 hours after the usual expectedtime.

Control of the natural termination of the sleep stage means the subjectis capable of naturally terminating the sleep stage of the sleep cycleat a particular desired time point. As such, control of initiation ofthe sleep stage includes the ability by the subject to wake up from thesleep stage of the sleep cycle at a particular time, where such time isearlier than the usual expected time, i.e., occurring before the usualexpected time, where such time is at least about 30 minutes to about 12hours, such as at least about 2 to about 10 hours, including at leastabout 3 to about 7 hours before the usual expected time. Control ofnatural termination of the sleep stage also includes the ability by thesubject to wake up from the sleep stage of the sleep cycle at aparticular time, where such time is later than the usual expected time,i.e., occurring after the usual expected time, where such time is atleast about 30 minutes to about 12 hours, such as at least about 2 toabout 10 hours, including at least about 3 to about 7 hours after theusual expected time.

Control of physiologically adapting to an environmentally imposed changein sleep cycle means the subject is required to sleep during a new timeschedule and that one or more physiological circadian rhythms (i.e.temperature rhythm, cortisol rhythm, melatonin rhythm, ACTH rhythm,etc.) is phase advanced or phase delayed in a desired direction to helpadapt to the imposed change in sleep cycle. As such, control ofmodifying one or more physiological circadian rhythms is determined bymeasuring a predetermined phase marker, where such time is earlier thanthe usual expected time, i.e., occurring before the usual expected time,where such time is at least about 30 minutes to about 12 hours, such asat least about 2 to about 10 hours, including at least about 3 to about7 hours before the usual expected time. As such, control of modifyingone or more physiological circadian rhythms is determined by measuring apredetermined phase marker, where such time is earlier than the usualexpected time, i.e., occurring before the usual expected time, wheresuch time is at least about 30 minutes to about 12 hours, such as atleast about 2 to about 10 hours, including at least about 3 to about 7hours before the usual expected time, where such time is at least about30 minutes to about 12 hours, such as at least about 2 to about 10hours, including at least about 3 to about 7 hours after the usualexpected time.

In some embodiments, where subjects are unable to initiate sleep or havedifficulty in initiating sleep, and where a phase advancement of thesleep cycle is desired, modulation of the circadian rhythm of a subjectcan be determined by evaluating the subject's ability to initiate sleepat a particular desired time. In such embodiments, an increase in thesubject's ability to initiate sleep at a particular desired time means,for example, the following: an increase in the value which is calculatedfrom the time that a subject sleeps divided by the time that a subjectis attempting to sleep (e.g., total time that a subject maintains sleepdivided by the total time the subject is attempting to initiate sleep),where such an increase is at least about 1.2 or more fold, such as atleast about 3 or more fold, including at least about 4, 6, 8 fold oreven higher, compared to a control; an increase in the total amount ofsleep, where such an increase is at least about 1.2 or more fold, suchas at least about 3 or more fold, including at least about 4, 6, 8 foldor even higher, compared to a control; a decrease in sleep latency (thetime it takes to fall asleep), where such a decrease is at least about1.2 or more fold, such as at least about 3 or more fold, including atleast about 4, 6, 8 fold or even higher, compared to a control; and adecrease in the time spent awake following the initial attempt toinitiate a sleep stage, where such a decrease is at least about 1.2 ormore fold, such as at least about 3 or more fold, including at leastabout 4, 6, 8 fold or even higher, compared to a control.

Other methods of evaluating modulation of the circadian rhythm of asubject include any testing methods involving interrogating the subjectand determining the effectiveness of the administration of the GRantagonist on modulating the subject's circadian rhythm. Such evaluationmay include first determining when the subject-desires to initiate thesleep stage of the sleep cycle prior to administration of the GRantagonist, and then determining when the subject was actually able toinitiate the sleep stage of the sleep cycle after administration of theGR antagonist.

Utility

The subject methods may be used in treating a subject suffering from asleep disorder, including for example, sleep problems associated withinsomnia, Circadian Rhythm Sleep Disorder, Delayed Sleep Phase Type,Circadian Rhythm Sleep Disorder, Advanced Sleep Phase Type, shift-workinduced sleep disorder, Circadian Rhythm Sleep Disorder, Jet Lag Type,Circadian Rhythm Sleep Disorder, Irregular Sleep-Wake Type, CircadianRhythm Sleep Disorder, Free-Running Type, short sleeper, long sleeperand the like. The methods may also be useful for treating HPA axisabnormalities in subjects suffering from a sleep-related breathingdisorder, such as obstructive sleep apnea, wherein such HPA axisabnormalities may contribute to insomnia. As such, the subject methodsare useful for treating subjects that are unable to initiate sleep andmaintain sleep, or unable to initiate sleep or maintain sleep, such assubjects suffering from insomnia, have difficulty in initiating sleep,such as subjects suffering from Circadian Rhythm Sleep Disorder, DelayedSleep Phase Type, or are subjects having difficulty in maintaining anawake state prior to a desired time of sleep initiation, such assubjects suffering from Circadian Rhythm Sleep Disorder, Advanced SleepPhase Type.

The present invention is further useful for subjects desiring anadaptation of the circadian rhythm to a different time schedule. Forexample, air travelers who rapidly cross two or more time zones may findtheir internal circadian clocks out of phase with the day/night cycle attheir destination, giving rise to the so-called “jet-lag” syndrome inwhich they suffer disruptions of their sleep patterns and diminishedattention span and alertness until their inner biological clocksgradually adjust to local time, will find use in modulating theircircadian rhythm in order to phase shift their circadian rhythm to adaptto the environmentally imposed desired sleep cycle In addition subjects,whose work schedules rotate among day shift, night shift and theso-called “graveyard” shift, and experience transient internal temporaldissociation or a lack of synchronization among various bodily rhythms,and consequent difficulty in adjusting to shift changes, will also finduse in modulating their circadian rhythm in order to phase shift theircircadian rhythm to adapt the environmentally imposed desired sleepcycle.

Thus, in one aspect the subject methods provides for treating a subject,where the subject suffers from a sleep disorder. As used herein the term“sleep disorder” refers to a disordered, interrupted or fragmented sleepcharacterized by events including, but not limited to, snoring, periodsof sleep apnea, narcolepsy, restless legs syndrome, sleep terrors, sleepwalking, and daytime somnolence. Such sleep disorders include, but arenot limited to, insomnia, Circadian Rhythm Sleep Disorder, Delayed SleepPhase Type, Circadian Rhythm Sleep Disorder, Advanced Sleep Phase Type,shift-work induced sleep disorder, Circadian Rhythm Sleep Disorder, JetLag Type, Circadian Rhythm Sleep Disorder, Irregular Sleep-Wake Type,Circadian Rhythm Sleep Disorder, Free-Running Type, short sleeper, longsleeper and HPA axis abnormalities associated with sleep apnea that mayinterfere with sleep or contribute to metabolic complications of sleepapnea and the like.

In another aspect the subject method provides for treating a subject,where the subject desires an adaptation to a differing time schedule,such as for example, inter-time zone travel induced sleep disturbance(Circadian Rhythm Sleep Disorder, Jet Lag Type) (i.e., jet-lag), shiftworkers' sleep disturbances, and the like. Sleep disorders and sleepdisturbances are generally characterized by difficulty in initiating ormaintaining sleep or in obtaining restful or enough sleep.

Treatment of a subject according to the subject methods can becorrelated to an enhancement of sleep quality of the subject. Anenhancement of sleep quality may be determined, for example, byexamining the following parameters in a subject after administration ofthe glucocorticoid receptor antagonist: an increase in the value whichis calculated from the time that a subject sleeps divided by the timethat a subject is attempting to sleep; a decrease in sleep latency (thetime it takes to fall asleep); a decrease in the number of awakeningsduring sleep stage; a decrease in the time spent awake following theinitial attempt to initiate a sleep stage; an increase in the totalamount of sleep; an increase the amount and percentage of REM sleep; anincrease in the duration and occurrence of REM sleep; a decrease in thefragmentation of REM sleep; an increase in the amount and percentage ofslow-wave (i.e. stage 3 or 4) sleep; an increase in the amount andpercentage of stage 2 sleep; a decrease in the number of awakenings,especially in the early morning; an increase in daytime alertness; andincreased sleep maintenance; a change in the distribution of REM and SWSduring the night (e.g., a change in the amount of SWS in the first halfof the night, or a change in the amount of REM in the second half of thenight). Wherein an increase in parameters noted above means an increaseof at least about 1.2 or more fold, such as at least about 3 or morefold, including at least about 4, 6, 8 fold or even higher, compared toa control, and wherein a decrease in the parameters noted above means adecrease of at least about 1.2 or more fold, such as at least about 3 ormore fold, including at least about 4, 6, 8 fold or even higher,compared to a control;

Thus, in one aspect the subject methods provide for treating a subjectfor a sleep disorder by administering to the subject an effective amountof a glucocorticoid receptor antagonist in a manner sufficient tomodulate the circadian rhythm of the subject. In some embodiments thesubject has already been identified as suffering form a sleep disorder.In other embodiments, the subject is first identified as suffering froma sleep disorder.

A sleep disorder is characterized by a difficulty in the ability of thesubject to initiate or maintain sleep or difficulty in obtaining restfulor enough sleep. Examples of sleep disorders include, but are notlimited to, insomnia, Circadian Rhythm Sleep Disorder, Advanced SleepPhase Type, Circadian Rhythm Sleep Disorder, Delayed Sleep Phase Type,shift-work induced sleep disorder, Circadian Rhythm Sleep Disorder, JetLag Type, Circadian Rhythm Sleep Disorder, Irregular Sleep-Wake Type,Circadian Rhythm Sleep Disorder, Free-Running Type, short sleeper, longsleeper and HPA axis abnormalities associated with sleep apnea that mayinterfere with sleep and the like.

Insomnia can be classified as transient, occurring only for a short termand, intermittent, occurring from time to time, and chronic, occurringon most nights and lasts a month or more. Symptoms of insomnia can bedifferent for each individual, and subjects suffering form insomniamight experience a variety of symptoms, such as: difficulty fallingasleep, which can mean lying in bed for up to an hour or more, includingtossing and turning; awakening during sleep and having trouble gettingback to sleep; awakening too early in the morning; feeling unrefreshedupon awakening; and daytime irritability, drowsiness, anxiety, and/ornonproductiveness.

Circadian Rhythm Sleep Disorder, Advanced Sleep Phase Type is a sleepdisorder in which the sleep cycle is advanced in relation to the desiredclock time, resulting in symptoms of compelling evening sleepiness, anearly sleep onset, and an awakening that is earlier than desired.Symptoms of Circadian Rhythm Sleep Disorder, Advanced Sleep Phase Typeinclude, for example, the following: inability to stay awake until thedesired bedtime or inability to remain asleep until the desired time ofawakening; a phase advance of the sleep cycle in relation to the desiredtime for sleep; and symptoms last for at least about 2 to 4 months,including at least about 3 months. When not required to remain awakeuntil the later bedtime, subjects suffering from Circadian Rhythm SleepDisorder, Advanced Sleep Phase Type will generally have a habitual sleepperiod that is of normal quality and duration, with a sleep onsetearlier than desired, awaken spontaneously earlier than desired, andmaintain stable entrainment to a 24-hour sleep-wake pattern.

Circadian Rhythm Sleep Disorder, Delayed Sleep Phase Type is a sleepdisorder in which the sleep cycle is delayed by at least about 1.5 ormore hours, including at least about 2 or more hours of the desiredbedtime. A subject suffering from such a sleep disorder will generallyhave difficulty awakening at the desired time. Symptoms of CircadianRhythm Sleep Disorder, Delayed Sleep Phase Type include, for example,the following: complaint of insomnia or excessive sleepiness; inabilityto fall asleep at the desired time; inability to wake up at the desiredtime; depression may be present; and such a sleep pattern lasts for atleast about 2 to 4 months, including at least about 3 months.

Short sleeper syndrome is characterized as a an individual whohabitually and spontaneously sleeps substantially less in a 24-hourperiod than is expected for a person in his or her age group, and doesnot have excessive sleepiness. Individuals that have short sleepersyndrome awaken spontaneously and have a daily total sleep time of lessthan about 65% to about 90%, such as about 70% to about 80%, includingabout 75% of the age-related norm.

Long sleeper syndrome is characterized as an individual who habituallyand spontaneously sleeps substantially more in a 24-hour period than isexpected for a person in his or her age group. Individuals that havelong sleeper syndrome have a daily total sleep time of more than about 1hour to about 4 hours, such as about 2 hours to about 3 hours, includingabout 2.5 hours of the age-related norm.

Obstructive sleep apnea is characterized as a cessation of breathingduring sleep that is caused by repetitive partial or completeobstruction of the airway by pharyngeal structures. Patients withobstructive sleep apnea often are overweight, snore loudly, and complainof daytime fatigue and sleepiness. The severity of the condition ismeasured by the number of apneas (i.e., cessations of airflow) orhypopneas (i.e., reductions in airflow) that cause sleep arousal.

Upper airway resistance syndrome characterized by a partial collapse ofthe upper airway resulting in increased resistance to airflow. Incontrast to Obstructive Sleep Apnea it is seen more often in women thanmen and snoring is the hallmark clinical symptom. Multiple sleepfragmentations are measured by short alpha EEG arousals but the syndromedoes not result in apneic and hypopneic events.

In such embodiments, the subject is administered an effective amount ofa glucocorticoid receptor antagonist in order to modulate the subject'scircadian rhythm, either by changing the phase or changing the shape ofthe waveform, thereby altering the subject's sleep.

Subjects suffering from a sleep disorder characterized by an inabilityto initiate and/or maintain sleep, such as insomnia, may be treatedaccording to the subject methods by administering to the subjects aneffective amount of a glucocorticoid receptor antagonist in order tomodulate the subjects' circadian rhythm waveform. In such embodiments,in which modifying the subject's circadian rhythm waveform is desired, aglucocorticoid receptor antagonist may be administered to the subjectsin a daily amount of between about 2 to about 20 mg per kilogram of bodyweight per day, such as in a daily amount of between about 2 to about 12mg per kilogram of body weight per day, including in a daily amount ofbetween about 8 to about 12 mg per kilogram of body weight per day. Thedose may be given in the morning. The dose may be given on either anintermittent or chronic basis.

Subjects suffering from a sleep disorder characterized by delayedability to initiate sleep, such as Circadian Rhythm Sleep Disorder,Delayed Sleep Phase Type, may be treated according to the subjectmethods by administering to the subjects an effective amount of aglucocorticoid receptor antagonist in order to modulate the subjects'circadian rhythm and phase advancing the subjects' sleep cycle. In suchembodiments, in which phase advancing the subjects' sleep cycle isdesired, a glucocorticoid receptor antagonist may be administered to thesubjects in a daily amount of between about 2 to about 20 mg perkilogram of body weight per day, such as in a daily amount of betweenabout 2 to about 12 mg per kilogram of body weight per day, including ina daily amount of between about 5 to about 9 mg per kilogram of bodyweight per day. The dose may be given in at a time of day designed toadvance the subject's circadian rhythm. The dose may be given on eitheran intermittent or chronic basis.

Subjects suffering from a sleep disorder characterized by an inabilityto maintain an awake state prior to desired time of sleep initiation,such as Circadian Rhythm Sleep Disorder, Advanced Sleep Phase Type, maybe treated according to the subject methods by administering to thesubjects an effective amount of a glucocorticoid receptor antagonist inorder to modulate the subjects' circadian rhythm and phase delay thesubjects' sleep cycle. In such embodiments, which phase delaying thesubjects' sleep cycle is desired, a glucocorticoid receptor antagonistmay be administered to the subject in a daily amount of between about 2to about 20 mg per kilogram of body weight per day, such as in a dailyamount of between about 2 to about 12 mg per kilogram of body weight perday, including in a daily amount of between about 5 to about 9 mg perkilogram of body weight per day. The dose may be given at a time of daydesigned to delay the subject's circadian rhythm. The dose may be givenon either an intermittent or chronic basis.

In another aspect, the subject methods provide for treating a subjectdesiring an adaptation of the subject's circadian rhythm to a differenttime schedule. In some embodiments, the subject methods may be used totreat air travelers who rapidly cross two or more time zones, such asthree to five time zones. In such embodiments, the subjects may findtheir internal circadian clocks out of phase with the day/night cycle attheir destination, giving rise to the so-called “jet-lag” syndrome,thereby suffering disruptions of their sleep patterns and diminishedattention span and alertness until the inner biological clocks of thesubjects' gradually adjust to local time. The subject methods may besuitable for treating subjects experiencing an inter-time zone travelinduced sleep disturbance (Circadian Rhythm Sleep Disorder, Jet LagType) desiring a phase advancement of the sleep cycle or a phase delayor the sleep cycle. In such embodiments, a glucocorticoid receptorantagonist may be administered to a subject in a daily amount of betweenabout 2 to about 20 mg per kilogram of body weight per day, such as in adaily amount of between about 2 to about 12 mg per kilogram of bodyweight per day, including in a daily amount of between about 5 to about9 mg per kilogram of body weight per day. The dose may be given in at atime of day designed to achieve a desirable advance or delay thesubject's circadian rhythm, to thereby help synchronize the subject tothe new time zone. The dose may be given on either an intermittentbasis.

In another embodiment, the subject method can be used to treat subjectswhose work schedules rotate among day shift, night shift and theso-called “graveyard” shift, and experience transient internal temporaldissociation or a lack of synchronization among various bodily rhythms,and consequently experience difficulty in adjusting to shift changes.The subject methods may be suitable for treating a subject experiencinga sleep disturbance resulting from shift work, where the subject desiresa phase advancement of the sleep cycle or a phase delay or the sleepcycle. In such embodiments, the subject may be administered aglucocorticoid receptor antagonist in a daily amount of between about 2to about 20 mg per kilogram of body weight per day, such as in a dailyamount of between about 2 to about 12 mg per kilogram of body weight perday, including in a daily amount of between about 5 to about 9 mg perkilogram of body weight per day. The dose may be given in at a time ofday designed to achieve a desirable advance or delay the subject'scircadian rhythm, to thereby help synchronize the subject to theirdesired schedule. The dose may be given on either an intermittent orchronic basis.

Kits

Also provided by the subject invention are kits for use in the subjectmethods described above. Kits for practicing the subject methods includea glucocorticoid receptor antagonist. In some embodiments, theglucocorticoid receptor antagonist includes a steroidal skeleton with atleast one phenyl-containing moiety in the 11-beta position of thesteroidal skeleton. In further embodiments, the phenyl-containing moietyin the 11-beta position of the steroidal skeleton is adimethylaminophenyl moiety. In yet further embodiments, theglucocorticoid receptor antagonist is mifepristone (i.e., RU486), RU009,RU044, ORG-34517, ORG-34850, or ORG-34116.

The kits may further include means for delivering the glucocorticoidreceptor antagonist to the subject, e.g. a syringe. The subject kitsfurther typically include instructions for carrying out the subjectmethods, where these instructions may be present on a package insertand/or the packaging of the kit. Such instructions may includeinstructions for using the glucocorticoid receptor antagonist fortreating a subject suffering from a sleep disorder such as insomnia,Circadian Rhythm Sleep Disorder, Delayed Sleep Phase Type, and CircadianRhythm Sleep Disorder, Advanced Sleep Phase Type, or phase shifting ahost's sleep cycle for treating a sleep disturbance resulting frominter-time zone travel (i.e., jet-lag) or shift-work. Such instructionmay further include information such as dosage and schedule ofadministration of the GR antagonist. These instructions may be presentin the subject kits in a variety of forms, one or more of which may bepresent in the kit. One form in which these instructions may be presentis as printed information on a suitable medium or substrate, e.g., apiece or pieces of paper on which the information is printed, in thepackaging of the kit, in a package insert, etc. Yet another means wouldbe a computer readable medium, e.g., diskette, CD, etc., on which theinformation has been recorded. Yet another means that may be present isa website address which may be used via the internet to access theinformation at a removed site. Any convenient means may be present inthe kits.

Experimental

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

EXAMPLE 1

Mifepristone for Treatment of Insomnia: Polysomnogram Measures

Recently, HPA axis hyperactivity has been reported in some forms ofinsomnia (Rodenbeck and Hajak, J Clin Psychiatry. 2001 June;62(6):453-63; Vgontzas et al., J Clin Endocrinol Metab. 2001 August;86(8):3787-94), without depression. A resetting of the HPA axis with aGR/PR antagonist has been described in psychiatric patients (Belanoff etal., Biol. Psych. Sep. 1, 2002; 52(5):386-92 ). An early report ofmifepristone in a healthy male reported significant worsening of sleepwith mifepristone (Wiedemann et al., Eur Arch Psychiatry Clin Neurosci.1992; 241(6):372-5). In this latter study, mifepristone was given in theafternoon and its longitudinal effect with time, after discontinuationwas not measured.

In this double-blinded placebo controlled study the longitudinal effectsof 5 days of a GR antagonist on HPA axis activity, melatonin and sleepin subjects with insomnia were measured. A 30 day repeated studyincorporating HPA axis activity (e.g., cortisol and ACTH) as well asmelatonin and sleep changes before, during, and after discontinuation oftreatment were measured. The design was formulated to additionallyestablish the temporal relationship between treatment and response,including determination of measurements two weeks after discontinuationof treatment. During the study all the subjects were put to bed at usualbed-time. The subjects were administered 600 mg of mifepristone per dayin the morning for five consecutive days. All subjects were permitted tosleep up until natural wake-up, limited to 7:45 to 8:00 am to allow forevaluation of increased natural total sleep time.

The results show that as a result of the administration of mifepristone,total sleep time (TST) increased (at 2 weeks post treatment) aftertreatment (FIGS. 1 and 2, Tables 1 and 2). This result was seen in PSGas well as during outpatient portion by prospective actigraphy measures.In addition, REM latency (RL) increased (most acutely during activetreatment), which is important with respect to the beneficial effect ofmifepristone administration in depression (FIGS. 1 and 2, Tables 1 and2) and in narcolepsy. Since REM latency changes with the phase of thecircadian core temperature rhythm, this shows that the circadian rhythmof temperature is delayed with treatment as well.

Sleep changes are associated with changes in ACTH, cortisol andmelatonin (acutely) and after treatment cessation. FIG. 4 showsincreased in the ratio of ACTH to cortisol post-treatment. Onepossibility is that this may represent decreased central CRH aftertreatment discontinuation with subsequent decreased direct brainstemnorepinephrine activation by brain CRH. TABLE 1 Change in PolysomnogramScores Treatment Group (Data set includes 5 double blinded treatmentpatients, and 5 double blinded placebo patients) 2 Wk Day 5 Tx Post TxRel to Rel to Baseline Tx Baseline Tx Tx Plac rel to Tx Plac rel toDelta Delta Plac Delta Delta Plac WASO 16.4 −21.45 37.85 −10.4 −18.257.85 (min) % S1 −38.34 −40.1 1.76 −39.19 −39.18 −0.01 % S2 6.44 2.54 3.9−2.68 2.37 −5.05 % S3 0.76 0.05 0.71 1.09 −0.36 1.45 % S4 −0.01 −0.430.42 −1.48 −0.5 −0.98 % REM −9.33 0.36 −9.69 1.63 0.32 1.31 NUAW 3.3−1.7 5 0 −1.5 1.5 TST (min) 17.05 20.8 −3.75 38.5 16.85 21.65 S3 Lat7.05 −12.3 19.35 −0.85 −23.95 23.1 (min) SEB −2.13 4.72 −6.85 2.57 3.91−1.34 (S2, S3, S4) SEC (S3, −7.3 0.15 −7.45 1.19 −0.83 2.02 S4) Rem Lat61.15 −38.45 99.6 −4 −43.5 39.5 (min) Rem p −0.826 −1.48 0.654 1.972−0.758 2.73 Arousal −0.35 −0.64 0.29 −0.85 0.24 −1.09 Index Arousal 0.94.7 −3.8 5.2 8.9 −3.7 SPT (min) 28.69 −0.7 29.39 28.1 −1.45 29.55Definitions:WASO = wake after sleep onset% S1 = percentage stage 1 sleep% S2 = percentage stage 2 sleep% S3 = percentage stage 3 sleep% S4 = percentage stage 4 sleep% REM = percentage REM sleepNUAW = number of awakeningsTST = total combined minutes of sleepS3 Lat (min) = latency to onset of stage 3 sleepSEB = total combined minutes of stage 2, 3 and 4 per total sleep minutesSEC = total combined minutes of stage 3, 4 per total sleep minutes% NEM = sum of % S3 and % S4Rem lat = minutes to first epoch of REM after sleep onsetREM density = # rapid eye movements per minutes of REM sleepArousal index = computerized measure high frequency bands/minutes sleep

TABLE 2 Average Polysomnogram Measures (Data set includes 5 doubleblinded treatment patients, and 5 double blinded placebo patients) RxPlacebo Rx Placebo Rx Placebo Ave Ave Ave Ave Ave Ave Rx T1 Plac Rx T2Plac Rx T3 Plac (n = 5) SD (n = 5) SD (n = 5) SD (n = 5) SD (n = 5) SD(n = 5) SD WASO (min) 66.95 24.22 83.35 69.71 83.35 42.34 61.90 37.4356.55 19.73 65.10 44.75 % S1 13.21 6.62 11.70 3.97 15.48 9.65 9.10 2.7214.63 6.40 10.02 2.07 % S2 53.82 7.63 49.20 8.82 60.26 11.60 51.74 6.0351.14 1.96 51.57 6.17 % S3 6.87 4.57 10.92 5.00 7.63 5.87 10.97 5.307.96 4.32 10.56 5.39 % S4 4.37 8.25 6.22 5.58 4.36 8.37 5.79 5.49 2.894.77 5.72 4.61 % REM 20.95 2.97 21.33 5.41 11.62 5.86 21.69 3.72 22.583.98 21.65 4.08 NUAW 24.30 10.47 25.30 10.90 27.60 10.39 23.60 18.4624.30 15.53 23.80 16.37 TST (min) 373.60 74.21 364.20 64.40 390.65 61.95385.00 21.34 412.10 58.34 381.05 26.64 S3 Lat (min) 17.75 14.49 39.4540.38 24.80 23.44 27.15 19.63 16.90 10.39 15.50 3.62 SE (S2, S3, S4)85.47 5.25 81.88 13.52 83.34 7.83 86.60 7.15 88.04 3.47 85.79 9.24 SE(S3, S4) 32.24 10.73 38.46 8.56 24.94 17.20 38.61 6.50 33.43 6.71 37.637.26 Rem Lat (min) 76.80 13.41 113.60 53.98 137.95 55.79 75.15 35.4072.80 21.69 70.10 28.48 Rem density 7.99 3.16 9.68 5.14 7.16 3.73 8.204.54 9.95 2.99 8.92 4.60 Arousal Index 16.60 5.31 17.28 5.98 16.25 3.5116.64 7.68 15.75 3.51 17.52 7.04 Arousal 104.80 39.64 101.30 27.06105.70 30.24 106.00 50.67 110.00 36.40 110.20 47.17 SPT (min) 440.5575.72 447.60 58.59 469.24 66.18 446.90 34.85 468.65 65.41 446.15 40.17

TABLE 3 Actigraphy Measures of TST Minutes and WASO Placebo Tx (n = 3)(n = 2) D7AV-smin 485.76 458.86 D7AV-waso 39.19 45.14 D20AV-smin 452.33448.40 D20AV-waso 28.13 51.70 D26AV-smin 468.33 473.08 D26AV-waso 32.2231.67

EXAMPLE 2

Mifepristone for Circadian Phase Shifting: Hormonal Measures

Further applications for administration of a glucocorticoid antagonistinclude shifting the circadian rhythm of healthy subjects in preparingfor or rapidly adjusting internal hormonal rhythms during inter-timezone travel (e.g., travel between eastern United States to westernUnited States) and for adapting to shift-work. For example, beneficialeffects include an acute phase delay in melatonin rhythm (as measured byDLMO), acute phase delay in cortisol and ACTH acrophase, and delay inREM latency, as well as a delay in temperature rhythm, since REM latencyis a marker for the phase of the temperature rhythm). This could behelpful in delaying one's internal circadian rhythms to better adapt tosleeping in later time zone (as would occur on traveling from East toWest) and thus help ameliorate the symptoms of jet lag,

In the study described in Example 1, melatonin was additionally measuredunder dim lights to measure changes in-its circadian rhythm with time.Melatonin is considered to be a somewhat pure marker for the timing ofthe phase of the body clock, or supra-chiasmatic nucleus (SCN).

FIG. 3 shows absolute values of the timing of melatonin in both thetreatment and placebo groups before and after 5 days of a GR antagonistas well as 2 weeks post-discontinuation These data shows a delay in DLMO(time at which melatonin reaches 20 pg/ml) in the GR antagonist groupafter 5 days treatment in the outpatient setting (which is consistentwith a delay in the timing of the SCN).

In a separate experiment, the longitudinal effects of 2 days of a GRantagonist on melatonin and HPA axis activity was measured in healthycontrol subject during an inpatient stay under constant routineconditions. In this repeated measure study design, a subject was given400 mg of mifepristone for two consecutive days as a test protocol.Salivary cortisol, ACTH and dim light melatonin were measured theevening/morning before and on the second evening/morning of medication.The design was created to include measures of the phase of night-timeDLMO as well as measures of the morning response of the HPA axis,including timing of cortisol acrophase. The design was formulated toascertain circadian phase shifting in a controlled setting with healthysubjects (as might occur in jet-lag or shift work). FIG. 5 shows theresults from a single subject during a test protocol. In addition to theexpected rise in cortisol, this study shows a delay in the timing of themorning acrophase of cortisol with treatment. Salivary melatonin sampleswere insufficient.

EXAMPLE 3

The Acute Effects of a Mineralocorticoid (MR) Agonist on NocturnalHypothalamic-Adrenal-Pituitary (HPA) Axis Activity in Healthy Controls

I. Methods:

A. Subjects

Nine healthy subjects were recruited from the community via flyers andinternet postings. Subjects responding to ads were phone screened todetermine if subjects met initial eligibility criteria. Those subjectsmeeting criteria underwent physical examination, screening labs(complete blood count, comprehensive metabolic panel, urine analysis,urine toxicology screen, TSH, FT4, serum pregnancy test) and EKG. A SCIDwas performed to rule out Axis I and Axis II DSM-TR pathology. At thetime of this analysis and manuscript preparation, 9 subjects have beenenrolled. Inclusion criteria were: (1) participants are age 18 to 85years old with a HAM-D score less than or equal to 5. Exclusion criteriawere: (1) personal history of Axis I or Axis II disorders; (2) activemedical problems; (3) abuse of drugs or alcohol in the 6 months prior tostudy; (4) use of additional prescription medications, street drugs oralcohol during the week before the study; (5) currently pregnant orlactating.

B. Study Design

This study is a two day repeated measures study of fludrocortisone'sacute effects on nocturnal HPA axis activity in healthy controls. Bloodsamples were taken every 30 minutes, beginning at 1600 to 2400 onDays 1. At 1400 on Day 2, all subjects were given 0.5 mg offludrocortisone. Blood was then resampled from 1600 to 2400 on Day 2 toassess nocturnal changes in HPA axis activity. Blood sampling was timedin the first half of the night to coincide with the time period ofgreatest activity of MR.

C. Statistics

Group averages, standard deviation and effect sizes are reported. Theprimary end-points were chosen to be mean cortisol and ACTH from 1600 to2400 when greatest mineralocorticoid receptor activity is expected. Todetermine the differential effects of fludrocortisone, data from allsubjects are pooled and the average values from 1600 to 2400 arecomputed. Next, mean values across this time interval are computedbefore (Time 1) and after (Time 2) fludrocortisone and the effect sizefor the difference between pooled mean values is computed.

II. Results:

A. Demographic Data

A total of 11 subjects met all inclusion/exclusion criteria andcompleted the study at the time of this interim study and analysis. Ofthese, 2 subjects were excluded from the analysis due to inadequate ACTHsamples. Of the 9 HC subjects, there were 3 males and 6 females. Theaverage age was 30.9 (10.8), ranging from 21 to 54.

B. Primary End-Points

Cortisol. Average cortisol levels versus time are given in FIG. 6. Shownare baseline cortisol from 1600 to 2400 on the nights immediately beforeand after afternoon administration of fludrocortisone. Table 4 givesmean and standard deviation for both time periods. Relative to baseline,treatment with 0.5 mg fludrocortisone decreased mean cortisol during theinterval from 1600 to 2400 from 3.99 ug/dl (1.283) to 1.77 ug/dl (1.38),for a difference of 2.19 ug/dl (p=0.003, effect size 1.65). Thisdecrease coincides with a lowering of cortisol and ACTH at the nadir.TABLE 4 Effect of Fludrocortisone on Mean Cortisol and ACTH ValuesDifference Standard AUC Standard (Tx − Baseline) AUC Baseline DeviationTreatment Deviation P value Effect Size Mean Cortisol 2.19 3.96 1.2831.77 1.38 0.003 1.65 (1600 to 2400) Mean ACTH 5.76 18.87 1.275 13.2 1.270.0000 4.46 (1600 to 2400) Mean Cortisol/ACTH 0.07 0.192 0.061 0.1240.084 0.0686 0.92 (1600 to 2400)

ACTH. Average ACTH levels versus time are given in FIG. 7. Shown arebaseline ACTH from 1600 to 2400 on the nights immediately before andafter afternoon administration of fludrocortisone. Again, Table 4 givesmean and standard deviation for both time periods. Relative to baseline,treatment with 0.5 mg fludrocortisone decreased mean ACTH during theinterval from 1600 to 2400 from 18.87 pg/ml (1.275) to 13.2 pm/ml(1.27), for a difference of 5.76 pg/ml (p<0.0001, effect size 4.46).

Post-Hoc Analysis Post-hoc analysis was performed to determine iffludrocortisone has a differential effect on the ratio of cortisol toACTH concentration. FIG. 8 shows average ratios versus time before andafter fludrocortisone. Mean of this ratio from 1600 to 2400 is computedagain: Relative to baseline, treatment with 0.5 mg fludrocortisonedecreased mean of the ratio cortisol/ACTH during the interval from 1600to 2400 from 0.192 (0.061) to 0.124 (0.084), for a net decrease of0.0686 (p=0.0058, effect size=0.92).

III. Discussion:

This pilot study evaluated acute effects of fludrocortisone on nocturnalHPA axis activity in healthy controls. Nocturnal HPA axis hyperactivityis associated with many conditions, including chronic insomnia(Rodenbeck and Hajak 2001; Vgontzas, Bixler et al. 2001; Buckley andSchatzberg 2005), depression and healthy aging (Born and Fehm 1998;Buckley and Schatzberg 2005). MR activity is greatest in the earlynocturnal sleep period (Spencer, Kim et al. 1998) and plays an importantrole in decreasing the nocturnal nadir of cortisol. Nocturnal cortisolmay be an indirect marker for nocturnal CRH activity.

Endogenous cortisol is an activator of MR and GR, and has greateraffinity for MR than GR (Reul and de Kloet 1985). It has thus beenassumed that MR's are fully occupied prior to GR's being occupied bycortisol. Along this traditional line of thinking, in a recent study ofhealthy and aged subjects. (Otte, Yassouridis et al. 2003), metyraponewas used to first unload glucocorticoid receptors and then the MRagonist fludrocortisone was administered. The nocturnal level ofcortisol subsequently decreased. The ability of an MR agonist, alone, toadditionally activate MR (above and beyond that from cortisol) andinhibit nocturnal HPA axis, to our knowledge, has not been reported.Herein, the MR agonist fludrocortisone was given without first“unloading” receptors with metyrapone and its effect on nocturnal HPAaxis activity was measured. We predicted that, although endogenouscortisol may have greater affinity for MR than GR in brain, a direct MRagonist additionally would activate MR, above that of endogenouscortisol, to inhibit HPA axis activity. This inhibition of HPA axisactivity would manifest in terms of decreased concentration of ACTH andcortisol. Since MR activity is greatest during the first part of thenight and around the time of the -nadir, only the first part of thenight was studied. We presumed that an MR agonist would inhibit HPA axisactivity via hippocampal inhibition of the paraventricular nucleus viathe nucleus basalis terminalis pathway. Alternatively, fludrocortisonemay directly alter adrenal sensitivity to ACTH at the level of theadrenal gland.

Results show 0.5 mg fludrocortisone decreases mean cortisol from 1600 to2400 with a large effect size of 1.65. Similarly, it also decreases meanACTH from 1600 to 2400 with a large effect size of 4.46. Furthermore,the ratio of cortisol/ACTH also decreases during the same period, withan effect size of 0.92. Both the decrease in cortisol and ACTH aresimilar to those reported when fludrocortisones was given after“unloading” mineralocorticoid receptors by pre-treating with metyrapone(Otte, Jahn et al. 2003).

That fludrocortisone acutely decreases nocturnal cortisol and ACTH,suggests that it may be an agent for decreasing brain hypothalamic CRH.Furthermore, it suggests that even though endogenous cortisol has agreater affinity for MR than GR, MR is not always fully activated and issubject to agonist effects. This finding is significant in that theability to alter HPA axis activity has useful clinical applications. Forexample, conditions associated with HPA axis hyperactivity includechronic insomnia, depression and healthy aging. Comorbid cognitivebenefits are expected as well, given the known associations betweenexcess glucocorticoid activity and cognitive function. Thus, an MRagonist such as fludrocortisone will have clinical applications in thesepopulations.

Another interesting effect is the trend towards a reduction in the ratioof cortisol/ACTH towards the nadir which may represent a decrease inadrenal sensitivity to ACTH. We know that adrenal sensitivity changesacross the 24 hour period, and is lowest at the time of the nocturnalnadir. Rat studies show that cortisol secretion is affected by a directautonomic connection from the suprachiasmatic nucleus (SCN) to theadrenal cortex (Buijs, Wortel et al. 1999) as well as by splanchnicinnervation of the adrenal gland (Ulrich-Lai, Arnhold et al. 2006). Inthe former case, cortisol is secreted without ACTH stimulation. In thelatter case, splanchnic innervation influences adrenal sensitivity toACTH. In both cases, the ratio of cortisol/ACTH is impacted. As such,the MR agonist fludrocortisone also decreases adrenal sensitivity toACTH, in addition to its apparent inhibitory effect on CRH and ACTH atthe level of the PVN. An alteration in adrenal sensitivity is consistentwith an earlier study suggesting that an MR antagonist increased adrenalsensitivity to ACTH (Young, Lopez et al. 1998).

In summary, to our knowledge, this is the first study of the MR agonistfludrocortisone on nocturnal HPA axis activity, without first unloadingthe receptors with metyrapone. The effect size for a net decreased inmean cortisol and mean ACTH, from 1600 to 2400, is large. This indicatesa significant impact of fludrocortisone on inhibiting nocturnal HPA axisactivity. Furthermore, the fact that fludrocortisone can inhibit theaxis without first depleting cortisol by pre-treatment with metyrapone,indicates that brain MR's are not fully occupied. The decrease incortisol per ACTH indicates a role on adrenal sensitivity as well. Theability to lower nocturnal HPA axis activity has many useful clinicalimplications, including its benefit in insomnia, depression, healthyaging and other disorders associated with enhanced HPA axis activation.

It is evident from the above results and discussion that the subjectinvention provides for highly efficient methods and compositions formodulating the circadian rhythm of a subject, which can be employed inthe treatment of individuals suffering from sleep disorders and inindividuals desiring to adapt to different time schedules. As such, thepresent invention represents a significant contribution to the art.

The preceding merely illustrates the principles of the invention. Itwill be appreciated that those skilled in the art will be able to devisevarious arrangements which, although not explicitly described or shownherein, embody the principles of the invention and are included withinits spirit and scope. Furthermore, all examples and conditional languagerecited herein are principally intended to aid the reader inunderstanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofpresent invention is embodied by the appended claims.

1. A method for modulating a circadian rhythm of a subject comprising:administering to the subject an effective amount of at least one ofglucocorticoid receptor antagonist, a CRH antagonist and an MR agonistto modulate the circadian rhythm in the subject.
 2. The method of claim1, wherein the modulating results in phase shifting the subject's sleepcycle.
 3. The method of claim 1, wherein the method is a method fortreating a subject for a sleep disorder.
 4. The method of claim 3,wherein the sleep disorder is insomnia.
 5. The method of claim 3,wherein the sleep disorder is Circadian Rhythm Sleep Disorder, DelayedSleep Phase Type.
 6. The method of claim 3, wherein the sleep disorderis Circadian Rhythm Sleep Disorder, Advanced Sleep Phase Type.
 7. Themethod of claim 1, wherein the method is a method for treatingObstructive Sleep Apnea.
 8. The method of claim 1, wherein the method isa method for treating inter-time zone travel induced sleep disturbance.9. The method of claim 1 wherein the method is a method for treatingCircadian Rhythm Sleep Disorder, Shift Work Type.
 10. The method ofclaim 1 wherein the method is a method for treating Narcolepsy.
 11. Themethod of claim 1, wherein said method comprises administering aglucocorticoid receptor antagonist to said subject.
 12. The method ofclaim 11, wherein the glucocorticoid receptor antagonist ismifepristone.
 13. The method of claim 11, wherein the glucocorticoidreceptor antagonist is selected from the group consisting of RU009 andRU044.
 14. The method of claim 1, wherein the subject is a mammal. 15.The method of claim 14, wherein the mammal is human.
 16. The methodaccording to claim 1, wherein said method is a method for modulatingphase shifting of a sleep cycle of a mammalian subject.
 17. The methodof claim 16, wherein the method is a method for treating a subject for aCircadian Rhythm Sleep Disorder, Shift Work Type.
 18. The method ofclaim 16, wherein the method is a method for treating a subject forinter-time zone travel induced sleep disturbance (Circadian Rhythm SleepDisorder, Jet Lag Type).
 19. The method according to claim 3, whereinsaid method further comprises identifying said subject suffering from asleep disorder.
 20. A kit comprising: a glucocorticoid receptorantagonist; and instructions for using the glucocorticoid receptorantagonist to treat a sleep disorder in a subject.